Bilayer structure which encapsulates multiple containment units and uses thereof

ABSTRACT

The present invention provides a bilayer structure for encapsulating multiple containment units. These containment units can attach or contain therapeutic or diagnostic agents that can be released through the bilayer structure. A suitable example of such a containment unit is a unilamellar or multilamellar vesicle.

[0001] This application is claiming the priority under 35 U.S.C. §119(e)of provisional application, U.S. Ser. No. 60/032,306, filed Dec. 2, 1996and is a continuation in part application of U.S. Ser. No. 08/980,332,filed Nov. 28, 1997 both of which are incorporated by reference.

[0002] This invention was made with Government support under NSF grantDMR-9123048 and NIH grant GM47334. The Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

[0003] Conventional drug delivery technology, which in the past hasconcentrated on improvements in mechanical devices such as implants orpumps to achieve more sustained release of drugs, is now advancing on amicroscopic and even molecular level. Recombinant technology hasproduced a variety of new potential therapeutics in the form of peptidesand proteins and these successes have spurred the search for newer andmore appropriate delivery and targeting methods and vehicles.

[0004] Microencapsulation of drugs within biodegradable polymers andliposomes has achieved successes in improving the pharmacodynamics of avariety of drugs such as antibiotics and chemotherapeutic agents. Forexample, unilamellar vesicles are currently used as drug deliveryvehicles for a number of compounds where slow, sustained release ortargeted release to specific sites in the body are desired. The drug tobe released is contained within the aqueous interior of the vesicle andrelease is achieved by slow permeation through the vesicle bilayer. Avariety of modifications of the unilamellar vesicle membrane have beenattempted, including polymerizing or crosslinking the molecules in thebilayer to enhance stability and reduce permeation rates, andincorporating polymers into the bilayer to reduce clearance bymacrophages in the bloodstream.

[0005] One example of such a vesicle structure is known as Depofoam.Depofoam is a multivesicular particle that is created by multipleemulsification steps. A defined lipid composition is dissolved in avolatile solvent. The dispersed lipids in solvent are vigorously mixedwith water to form a first emulsion, designated a solvent continuousemulsion.

[0006] This first emulsion is then added to a second water/solventemulsion and emulsified to form a water in solvent in water doubleemulsion. The solvent is removed from the mixture resulting in discretefoam-like spherical structures consisting of bilayer separated watercompartments. The minimum size of these structures is about 5-10microns. Depofoam does not include a distinct bilayer structure thatencapsulates the multivesicular particles, i.e., there are noindividual, distinct interior vesicles. Therefore, the interiorcompartment must share bilayer walls.

[0007] Liposomes are sealed, usually spherical, either unilamellar ormultilamellar vesicles which are capable of encapsulating a variety ofdrugs. Liposomes are the most widely studied vesicles to date and theycan be formulated with a variety of lipid types and compositions thatcan alter their stability, pharmacokinetics and biodistribution. A majordisadvantage of both multilamellar and unilamellar liposomes as deliverysystems is their size, which prevents them from crossing most normalmembrane barriers and limits their administration to the intravenousroute. In addition, their tissue selectivity is limited to thereticuloendothelial cells, which recognize them as foreignmicroparticulates and then concentrates the liposomes in tissues such asthe liver and spleen.

[0008] Polymers have also been used as drug delivery systems. Theygenerally release drugs by (1) polymeric degradation or chemicalcleavage of the drug from the polymer, (2) swelling of the polymer torelease drugs trapped within the polymeric chains, (3) osmotic pressureeffects, which create pores that release a drug which is dispersedwithin a polymeric network, and/or (4) simple diffusion of the drug fromwithin the polymeric matrix to the surrounding medium.

[0009] With the success and drawbacks of these microencapsulationvehicles, today the challenge is to produce better and more efficientmicroencapsulation vehicles to enhance drug delivery. The presentinvention is directed to meeting that challenge.

SUMMARY OF THE INVENTION

[0010] The present invention provides a bilayer structure forencapsulating multiple containment units, e.g., polymer containmentunits. Containment units can attach or contain therapeutic or diagnosticagents that can be released through the bilayer structure. A suitableexample of such a containment unit is a unilamellar or multilamellarvesicle.

[0011] The invention provides compositions comprising the bilayerstructure of the invention and multiple containment units. Preferably,the containment units are aggregated.

[0012] In one embodiment of the invention, these compositions includevesosomes. Vesosomes have a bilayer structure that encapsulates multiplecontainment units in the form of vesicles. Generally, the multiplevesicles are aggregated. Vesicles can be unilamellar or multilamellar.

[0013] The vesicles can either be of similar size and composition or ofvaried size and composition. Preferably, each vesicle is attached toanother (or aggregated) via ligand-receptor or antibody-antigeninteractions.

[0014] Further, the encapsulating bilayer structure can attach to theaggregated vesicles via ligand-receptor interactions. By optimizing boththe exterior bilayer structure and the interior vesicle compositions,the size and size distribution of the interior vesicles, the overallsize of the vesosome, the nature of the attachments of the vesicles, andthe type of additives to the outer bilayer (such as polymers or specificrecognition sites), an extremely versatile drug delivery system can bedeveloped for a variety of applications.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 is a freeze-fracture transmission electron micrograph of atypical vesosome prepared upon mixing cochleated cylinders with sizedvesicle aggregates (at a 1:1 mole ratio) prior to the addition of EDTA.There is only one outer bilayer, and the interior vesicles appear to bespecifically aggregated.

[0016]FIG. 2 is a freeze-fracture transmission electron micrograph of atypical vesosome prepared upon mixing cochleated cylinders with activesized aggregates (at a 1:1 mole ratio) after addition of EDTA. Again,there is only one outer bilayer, and the interior vesicles appear to bespecifically aggregated. The two figures differ only in that EDTA hasbeen added to the second sample to chelate the remaining calcium.

[0017]FIG. 3 is a schematic representation of one embodiment forvesosome production.

[0018]FIG. 4 is a freeze-fracture TEM view of a vesosome.

[0019]FIG. 5 is a schematic diagram of the processes set forth Examples1 and 2.

[0020]FIG. 6 is a photograph showing stable, small aggregates formed byadding streptavidin to biotin-labeled vesicles at a ratio of about 2surface accessible biotins to streptavidin, this corresponds to a totalmole ratio of about 4 biotins per streptavidin.

[0021]FIG. 7 is a line graph showing that vesicle aggregation andproduction continued indefinitely, producing multi-micron sizedaggregates that flocculated.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0022] As used in this application, the following words or phrases havethe meanings specified.

[0023] As used herein, the term “containment units” means any structurehaving space that can be occupied by an agent such as a therapeutic,diagnostic, or cosmetic agent. Typically, the structure is spherical butis not necessarily so.

[0024] As used herein the term “vesosome” is an aggregate of unilamellaror multilamellar vesicles encapsulated within a distinct bilayerstructure. The interior vesicles can be of a single size and membrane orinterior composition or number of layers, or of varied sizes and/ormembrane or interior composition or number of layers. These parameterscan be controlled during the assembly process described herein.

[0025] As used herein “uniform size” means of approximately similarsize. It does not necessarily mean vesicles having an identical size.

[0026] In order that the invention herein described may be more fullyunderstood, the following description is set forth.

COMPOSITIONS OF THE INVENTION

[0027] The invention provides a bilayer structure for encapsulatingmultiple containment units.

[0028] The present invention further provides a vesosome having thebilayer structure of the invention that encapsulates multiple vesicles.Generally, the vesicles are aggregated within the bilayer structure (S.Chiruvolu et al. (1994) Science 264:1753). The interior vesicles can beof a single size and membrane or interior composition, or of variedsizes and/or membrane or interior composition. The interior vesicles caneither be unilamellar or multilamellar. These parameters can bedetermined during the assembly process described below. The totaldimensions of the vesosome can be controlled from about 0.05 micronto >5 micron. The vesosome can incorporate a variety of water or lipidsoluble drugs or other solutes within the interior vesicles, or withinthe exterior capsule, or both. These drugs can then permeate slowlythrough the interior and exterior bilayers, providing a controlled, slowrelease of drugs over time. Aggregation can be effected byligand-receptor or antibody-antigen interactions. Other aggregationmeans are possible. The vesosome of the invention is submicroscopic insize. Further, in accordance with the practice of the invention, themultiple vesicles are of uniform size. Alternatively, the multiplevesicles are of dramatically different sizes.

METHODS FOR MAKING AND USING COMPOSITIONS OF THE INVENTION

[0029] The invention further provides a method for encapsulatingmultiple vesicles within an outer membrane. This method comprisesobtaining aggregated multiple vesicles in a solution. Cochleatedcylinders are then added to the solution [Papahadjopoulos, D., et al.,(1974) Biochim. Biophys. Acta, 401, 317-335; Papahadjopoulos, D., etal., (1975) Biochim. Biophys. Acta, 394, 483-491; Papahadjopoulos, D.,et al., (1976) Biochim. Biophys. Acta, 448, 265-283].

[0030] The aggregated vesicles and cochleated cylinders are mixed in thesolution under suitable conditions so that the cochleated cylinderstransform to create the bilayer structures of the invention whichencapsulates the aggregated multiple vesicles.

[0031] In accordance with the practice of the invention, the interiorvesicles can include a therapeutic agent. Alternatively, the interiorvesicles can include a diagnostic agent. The interior vesicles caninclude a reactive agent, so as to create new compounds in situ. Methodsfor including agents within the containment units (e.g., vesicles) arewell known because methods for including such agents into liposomes arewell known (CA 1314209; DE 3880691; GB 9605915; DE 4402867).

[0032] Suitable therapeutic agents include, but are not limited to, thefollowing.

[0033] The therapeutic agent can include antimicrobial agents such asantibiotics, antifungal, and antimycobacterial drugs. Examples ofantibiotics include, but are not limited to, amikacin, kanamycin B,amphomycin, bacitracin, bicyclomycin, capreomycin, polymyxin E,cycloserine, chloramphenicol, dactinomycin, erythromycin, gentamicin,gramicidin A, penicillins, rifamycins, streptomycin, and tetracyclines.

[0034] The therapeutic agent can be a drug acting at synaptic andneuroeffector junctional sites. Examples include neurohumoraltransmitters, cholinergic agonists, anticholinesterase agents,antimuscarinic drugs, agents acting at the neuromuscular junction andautonomic ganglia, catecholamines, sympathomimetic drugs, and adrenergicreceptor antagonists.

[0035] Alternatively, the therapeutic agent can be a drug acting on theCNS. Examples include antipsychotic drugs, neuroleptic drugs, tricyclicantidepressants, monoamine oxidase inhibitors, lithium salts, andbenzodiazepines.

[0036] Additionally, the therapeutic agent can be a drug that reducesinflammation. Examples include antagonists of histamine, bradykinin,5-hydroxytryptamine; lipid-derived autacoids; methylxanthines, cromolynsodium; and analgesic-antipyretics.

[0037] The therapeutic agent can be a drug that affects renal functionand electrolyte metabolism. Examples include diuretics and inhibitors oftubular transport of organic compounds.

[0038] The therapeutic agent can be a drug that affects cardiovascularfunction. Examples include renin and angiotensin; organic nitrates,calcium-channel blockers and beta-adrenergic antagonists;antihypertensive agents, digitalis, antiarrhythmic drugs, and drugs usedin the treatment of hyperlipoproteinemias.

[0039] Suitable diagnostic agents include, but are not limited to,radiolabels, enzymes, chromophores and fluorescers.

[0040] In accordance with the practice of the invention, the aggregatedmultiple vesicles of step (a) of the method and the cochleated cylindersof step (b) of the method can be mixed in a suitable ratio, e.g., a 1:1ratio. Other ratios are possible.

[0041] The present invention provides a method for delivering atherapeutic agent to a wound site. This method comprises introducing thevesosome of the invention to the wound site. This is done underconditions so that the therapeutic agent is released from the vesosometo the wound site. Alternatively, the method also include delivering anagent to an intended site for cosmetic, veterinary, and otherapplications requiring slow release of a particular agent (CA 1314209;DE 3880691; GB 9605915; DE 4402867).

[0042] Introduction of the vesosome to the wound site can be effected byvarious methods. For example, the vesosome can be introduced byintramuscular injection, intravenous injection, oral administration,pulmonary adsorption, rectal administration, subcutaneous injection,sublingual administration, or topical application. Other methods areadministration methods are possible and well known in the art.

[0043] The most effective mode of administration and dosage regimen forthe molecules of the present invention depends upon the severity andcourse of the disease, the subject's health and response to treatmentand the judgment of the treating physician. Accordingly, the dosages ofthe molecules should be titrated to the individual subject.

[0044] The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m² of surface area is described byFerrite, E. J., et al. (Quantitative Comparison of Toxicity ofAnticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man. CancerChemother, Rep., 50, No.4, 219-244, May 1966).

[0045] Adjustments in the dosage regimen can be made to optimize theresponse. Doses can be divided and administered on a daily basis or thedose can be reduced proportionally depending upon the situation. Forexample, several divided doses can be administered daily or the dose canbe proportionally reduced as indicated by the specific therapeuticsituation.

[0046] The invention further provides a method for obtaining aggregatedvesicles having a uniform size. As used herein, “uniform size” meanshaving about the same, but not necessarily having identical, size.

[0047] This method comprises filtering aggregated vesicles havingvarying sizes through multiple membranes under pressure. By doing so,the vesicle aggregates so filtered have substantially uniform size. Forexample, the vesicles so extruded can have a size ranging from 0.05-5μm.

[0048] The vesicles are filtered through two membranes, although this isnot essential to the process. Generally, the membranes have filter poresof uniform size.

Possible Modifications and Variations

[0049] As mentioned, the detailed composition and size of the interiorvesicles is not important to the process. Other types of vesiclepreparation methods can be used, from (1) chemical preparations such asreverse phase evaporation, detergent dialysis, pH jump, (2) othermechanical treatments such as ultrasonication, and (3) spontaneousvesicle preparations which lead directly to equilibrium vesicles(without special treatments). Unencapsulated drug product can be removedat any stage of this process by various dialysis techniques, ionexchange, chromatography, filtration or centrifugation.

[0050] The aggregation of the vesicles can also be accomplished by avariety of ligand-receptor interactions, via antigen-antibody, or viachemical crosslinking agents that mimic ligand-receptor interactions.

[0051] Aggregate sizing can be accomplished by other methods such as (1)quenching the aggregation (adding another ligand that binds to thereceptor, preventing it from cross-linking more vesicles), (2) usingcharged vesicles that will aggregate at a slower rate due to enhancedelectrostatic repulsion between the vesicles and (3) altering thestoichiometric ratio of the ligand to receptor, which also can lead to aslower, controlled aggregation. Monodisperse aggregate sizes can beprepared by removing excess freely floating vesicles or small aggregatesby various centrifugation or dialysis techniques. Finally, polymers suchas polyethylene glycol linked to lipids can be incorporated into theexterior membrane to sterically stabilize the vesosome againstaggregation and/or clearance by macrophages. The various processes canbe optimized for a particular drug release or other application.

[0052] There are also different methods of forming the exterior membranein addition to the method described above. The simplest and most generalis to use a layer of large vesicles attached to the aggregated vesicles,then osmotically stress these exterior vesicles to “pop” them, therebyforming a continuous encapsulating membrane in the process. A secondmethod is to use large, flaccid vesicles to engulf the aggregatedvesicles in a type of endocytosis process, again mediated by ligandreceptor or similar interactions.

[0053] Advantages of the invention: The benefits of the vesosome oversingle walled vesicles used for drug delivery is that important membranefunctions can be divided among two or more membranes rather than one.For example, the permeation rate, the membrane charge, specificrecognition molecules, steric stabilizers, membrane rigidity and phasetransition temperatures all play a role in the optimization of a drugdelivery vehicle.

[0054] With the vesosome structure, these often incompatible attributescan be divided among the various membranes. The exterior membrane canincorporate steric stabilizer molecules such as polyethylene glycol, orspecific recognition sites such as ligand or specific receptors. Theinterior vesicles can be made of various sizes and compositions tooptimize the permeation of drugs from the vesosome. The interiorvesicles within the vesosome can be of different composition or sizefrom each other as well to optimize delivery of multiple drugs, orprolong delivery over time.

[0055] Building the vesosome takes advantage of several new featuresincluding:

[0056] 1) Specific aggregation of vesicles via ligand-receptorinteractions (Chiruvolu et al., 1994)

[0057] 2) Sizing the vesicle aggregates via extrusion

[0058] 3) Encapsulating the vesicles within a second membrane. As far aswe have been able to determine from the scientific literature, this isthe first time vesicles have been encapsulated within another bilayer bya controlled and reproducible process [Cevc, G. and Marsh, D. (1987)Phospholipid Bilayers: Physical Principles and Models, Wiley, New York;Lasic, D. D. (1993) Liposomes: From Physics to Applications, Elsevier,Amsterdam].

[0059] The greatest benefit to this step-wise process of construction isthe great flexibility it allows in optimizing bilayer composition,aggregate size, etc. Also, as many of the steps in the process arespontaneous self-assembly steps, they are especially simple and onlyinvolve mixing one or more solutions. As a result, these steps are quiteefficient and easy to scale up.

[0060] The following example is presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The example is not intended in any way to otherwise limit thescope of the invention.

EXAMPLE1

[0061] In this example, the preparation of a vesosome is essentially atwo-step process (FIGS. 3 and 5). The first step is creating acontrolled-size vesicle aggregate. The second step is encapsulating thevesicle aggregate within an outer membrane. Here we provide a specificexample of the techniques used to create the vesosomes shown in FIGS. 1and 2 above. In each case, the specifics of the lipids and crosslinkingagents, the size distributions, etc., used are only representative andcan be optimized to suit the application.

Vesicle Preparation

[0062] The vesicles can be made from a variety of phospholipids,cholesterol, fatty acids, etc. as needed. To create the vesosomes shownin FIGS. 1 and 2, 150 mg of dilauroylphosphatidylcholine (DLPC) (AvantiPolar Lipids, Alabaster, Ala.) and 0.4 mg ofbiotin-X-dipalmitoylphosphatidylethanolamine (B—DPPE) (Molecular Probes,Eugene, Oreg.) were mixed together in a 2 dram sample vial in chloroform(the B—DPPE was present at 0.163 mole % of total lipid in solution) tothoroughly mix the lipids. The chloroform was removed by evaporationunder vacuum. 5 mL of aqueous buffer/salt/azide solution (100 mM NaCl,50 mM TES, and 0.02 wt % NaN₃ balanced to pH 7.2) was added to the driedlipid to create a solution of 30 mg/mL total lipid. The sodium azide isused as a preservative, and is not necessary for the process.

[0063] After fully hydrating the lipids, the resultant solutionconsisted of multilamellar vesicles (MLVs). Unilamellar vesicles wereformed from the MLV's by a mechanical extrusion technique [Mayer, E.(1985) J Microsc. 140, 3-15]. The MLV'solution was repeatedly (1) frozenin a liquid nitrogen (T=−190° C.) bath for 30-60 seconds, then (2)immediately melted in a 50-60° C. water bath. This process disrupts themultilamellar structure of the vesicles and leads to the formation oflarge unilamellar vesicles (LUVs; polydisperse, up to a few microns insize). The solution is then allowed to cool to room temperature (25°C.). The LUV suspension is then put through 8-12 high pressure(approximately 50 psi dry nitrogen) extrusion cycles by filtering thesolution within an Extruder (Lipex Biomembranes, Vancouver, BC, Canada)through two stacked Nuclepore filters of pore diameter 0.1 μm. Thisprocess produces a 30 mg/mL monodisperse population of unilamellarvesicles (ULVs) approximately 100 nm in diameter. These vesicles consistof DLPC and B—DPPE, with B—DPPE being present in the bilayer at 0.163mole %. The biotin ligand is oriented away from the bilayer (in the samedirection as the headgroups). This creates a vesicle which has severalligands protruding from the both the interior and exterior surface. Thissolution of vesicles is then allowed to equilibrate for at least a fewhours. Although metastable, these ULVs remain freely suspended forseveral weeks without reverting to their equilibrium MLV structure.

Vesicle Aggregate Preparation

[0064] To aggregate the vesicles, an aqueous dispersion of streptavidinmolecules (mol. wt. 60,000 g/mol) in the same buffer solution is addedto the extruded vesicles. In this example, 3.94 mg of streptavidin(Molecular Probes, Eugene, Oreg.) is measured and mixed with 6.24 mL ofthe TES/NaCl/azide buffer solution to create a 0.63 mg/mL streptavidinsolution. 1.0 mL of streptavidin solution is added to a vial containing2.0 mL of the DLPC/B—DPPE ULV suspension. The overallbiotin-streptavidin mole ratio for this system is about 15:1, however,the ratio of exposed biotin (biotins on the outer vesicle monolayer) tostreptavidin is about 8:1. Since there are four identical binding sitesof streptavidin available for binding, the ratio of exposed biotins tobinding sites is 2:1. Within an hour, the 20 mg/mL ULV/streptavidinsuspension changes color from clear and bluish to opaque and cloudy,indicating that much larger particles are being formed, i.e., thevesicles are aggregating. This vesicle aggregation scheme does notappear to stress or rupture the individual vesicles. This process wasdeveloped in our laboratory [Chiruvolu, S., et al., (1994) Science 264,1753-1756].

Controlled-Size Vesicle Aggregates Preparation

[0065] Vesicle aggregate sizing is done by extruding the large vesicleaggregates through two stacked Nuclepore filters of pore diameter 1.0μm; this extrusion is essentially identical to the extrusion step in ULVproduction, except the pore size is larger. This produces a dispersionof vesicle aggregates with sizes ranging from 0.3-1.0 μm. Once formed,the sized vesicle aggregates are stable for weeks and experience minimalre-aggregation or re-dispersion.

Encapsulating Sized Aggregates

[0066] To encapsulate the vesicle aggregate, we take advantage ofmicrostructures common to negatively charged lipids in the presence ofcalcium ions. Cochleated cylinders are multilamellar lipid tubulesformed spontaneously by certain negatively charged phospholipids in thepresence of calcium ions. Ca²⁺is know to induce the adhesion, fusion andcollapse of bilayers containing large proportions of the anionicphospholipid phosphatidylserine (PS) [Papahadjopoulos, D., et al.,(1974) Biochim. Biophys. Acta, 401, 317-335; Papahadjopoulos, D., etal., (1975) Biochim. Biophys. Acta, 394, 483-491; Papahadjopoulos, D.,et al., (1976) Biochim. Biophys. Acta, 448, 265-283]. These dehydratedmultilamellar structures have been synthesized in our laboratory usingsimilar techniques reported in the literature as discussed below. Wehave independently confirmed the presence of cochleated cylinders in ourexperiments by FF-TEM.

DOPS Unilamellar Vesicle Preparation

[0067] Vesicles, composed of 1,2 dioleoylphosphatidylserine (DOPS;AVANTI Polar Lipids, Inc., Alabaster, Ala.) and containing small amountsof B—DPPE (Molecular Probes, Inc., Eugene, Oreg.) were made asprecursors to cochleated cylinders through similar methods as describedabove. Briefly, 50 mg of lyophilized DOPS (61.7 μmoles) was dissolved in5 mL of Chloroform with 0.1 ml of B—DPPE solution [9.8×10⁻⁸ mole B—DPPE]to give a mole fraction of B—DPPE of 0.0016. The chloroform wasevaporated under dry nitrogen and the lipid vacuum dried to removeexcess solvent. The dried, mixed lipids were then hydrated (orresuspended) in 5 mL aqueous buffer solution as described above,yielding a solution with DOPS (MW 810 g/mol) concentration of 10 mg/mL(12.3 mM) and a B—DPPE (MW 1019 g/mol) concentration of 0.02 mg/mL (0.02mM).

[0068] After dispersing the lipid by vortexing, we allowed equilibrationof the solution at 37° C. for 24 hours. The multilamellar vesiclesolution was taken through several freeze-thaw cycles prior to sizing byhigh pressure extrusion through Nuclepore 0.1 μm polycarbonatemembranes. The sized vesicles were allowed to equilibrate at 25° C.prior to the addition of Ca²⁺.

Ca²⁺Solution Preparation

[0069] Solutions containing millimolar quantities of free Ca²⁺wereprepared using anhydrous CaCl₂ salt (Sigma Chemical Co., St. Louis, Mo.)and the standard buffer solution. Previous experiments by our laboratoryrevealed that the concentration of Ca²⁺in solution required to inducefusion between small unilamellar vesicles of DOPS be greater than 2.0mM. A 6.0 mM CaCl₂ buffer solution was prepared for use in theseexperiments.

Cochleated Cylinders Preparation

[0070] Equal 1 mL volumes of the DOPS/B—DPPE vesicle solution (10 mg/mL)and the 6 mM CaCl₂ buffer solution were measured using two 1000 μLHamilton Gas-Tight™ syringes. The two solutions were simultaneouslydispensed into a clean, dry 3-dram vial, where they rapidly mixed toform a solution with a DOPS concentration of 5 mg/mL (6.2 mM), a B—DPPEconcentration of 0.01 mg/mL (0.01 mM) and a CaCl₂ concentration of 3 mM.

[0071] Immediately upon mixing, the turbidity of the solution increased.Aggregation, fusion and collapse of the DOPS/B—DPPE vesicles—andtransition into cochleated cylinders—began immediately.

[0072] Streptavidin (Molecular Probes, Inc., Eugene Oreg.) was dissolvedin the standard buffer for a solution with a concentration 0.63 mg/mL(1.0×10⁻⁸ mol/mL). 35 μL of the streptavidin solution was injected into1 mL of the cochleated cylinder solution to activate the cylinders. Theproduct was gently mixed and allowed to equilibrate for 24 hours.

Vesosome Preparation

[0073] We have now described how to prepare the two precursor solutions(in identical buffers) needed for vesosome production. First, we have asolution of active vesicle aggregates (with some active freely floatingvesicles). Second, we have a solution of active cochleated cylinders(with likely some freely floating streptavidin).

[0074] We have employed two different mixing ratios of the two precursorsolutions that produce the vesosome solutions. To briefly describe, onemixture is prepared such that the ratio of the number of moles of DLPClipids to DOPS lipids equals one. The second mixture is prepared suchthat the ratio of the approximate number of sized vesicle aggregates(taking into account the freely floating vesicles) to the approximatenumber of cochleated cylinders equals one. In the latter case, we areattempting to match at least one aggregate with one cylinder. In theformer case, we are ensuring that there are plenty of aggregates to getencapsulated.

[0075] In the mole-match case, 1.0 mL of the 5 mg/mL (DOPS) activecochleated cylinders/streptavidin solution is added to the 20 mg/mL(DLPC) 0.190 mL of active sized vesicle aggregates simultaneously. Thatis, 6.2 μmol of DOPS molecules is mixed with 6.2 μmol of DLPC molecules.Upon mixing, the solution turned from chunky, crystal-like structuresconsistent with suspensions of cylinder solutions to more opaque andless chunky.

[0076] In the number-match case, 1.0 mL of the 5 mg/mL (DOPS) activecochleated cylinders/streptavidin solution is added to the 20 mg/mL(DLPC) 0.040 mL of active sized vesicle aggregates simultaneously. Thatis, 6.2 μmol of DOPS molecules is mixed with 1.3 μmol of DLPC molecules.Therefore, in the mole-match case, there are about 5 times as manyaggregates as in the number-match case. Again, upon mixing, the natureof the solution changed.

Freeze-fracture TEM Results

[0077] Aliquots for freeze-fracture sample preparation were taken fromboth the mole-match and number-match solutions one day after mixing thecylinders and aggregates.

[0078] In general, FF-TEM revealed that most of the structures presentin either the mole-matched or number-matched solution were LUVs (1-5μm). Very few cylinders were observed. There did appear to be someunencapsulated sized aggregates as well a high concentration of freevesicles (100 nm). Actual concentrations or numbers are not available.

[0079] There did exist several vesosomes, as shown in FIG. 1. Theinterior aggregated vesicles (1) do appear to resemble the aggregatedvesicles in both size (˜0.5 μm) and aggregation state (dense andcompact) prior to mixing the solutions and (2) are approximately 100 nmin size. These features indicate that the vesicles are indeed the DLPCvesicles and not DOPS vesicles which have managed to remain even afterCa²⁺addition.

[0080] There are, however, a few larger vesicles present within, andthese may simply be larger DLPC ULVs or ULVs that have been formed bythe fusion of several DLPC ULVs.

[0081] Also, note that the vesosome has a single bilayer encapsulatingthe entire vesicle aggregate, consistent with the “unrolling” of acylinder.

[0082] A general encapsulation process could occur as follows: aftermixing the solutions, an active aggregate approaches an active cylinderand binds to its surface by at least one biotin-streptavidininteraction. The aggregate proceeds to bind in several places until thebinding force overcomes the force necessary to keep the cylinder wound.As the cylinder begins to unwind, the interior regions of the cylinder,now exposed to the aqueous solution, continue to bind around theaggregate until the cylinder unravels completely around the aggregate asif forced by the presence of EDTA.

[0083] Next, 0.44 mL of 5 mM EDTA solution (in the same buffer) wasadded to 0.5 mL of the mole-matched cylinder-aggregate mixture. Thecloudy, opaque solution immediately turned grayish and more transparent.This cylinder-aggregate mixture consisted of approximately 4.2 mg/mLDOPS (cylinders) and 3.2 mg/mL DLPC (aggregates). The amount of EDTAadded was in excess of the amount necessary to completely bind all ofthe available calcium ions and therefore cause unraveling of thecylinders. Also, 0.5 mL of the 5 mM EDTA solution was added to thenumber-matched mixture. Again, the solution changed to grayish andtransparent. This number-matched mixture consisted of 4.8 mg/mL DOPS and0.8 mg/mL DLPC. Again, excess EDTA was added. Aliquots of each of thesesolutions were also taken after approximately five hours of incubationfor freeze-fracture sample preparation.

[0084] FF-TEM again revealed that each of these solutions contained verymany LUVs (1-5 μm), as is expected in solutions in which EDTA has beenadded to cylinders. Also, there were several ULVs but no cylinders.

[0085] Vesosomes again were present in the number-match solution. FIG. 2shows a typical vesosome seen in these solutions. Note, again, thatinterior vesicles appear to be aggregated as in the precursor solutions.However, there are also some very large vesicles, probably unraveledDOPS vesicles, which have become encapsulated as well. The number ofvesosomes relative to the number of LUVs in these solutions does notseem to vary between the pre- and post-EDTA solutions, however, thereseem to be more of them in the mole-matched solutions. This may indicatethat the more active particles added to solution increases the chancesfor a vesosome to form.

[0086] It should be noted that the vesosome structures were not presentin either of the precursor solutions. The solutions of cylinderssaturated with streptavidin did not show any unusual characteristics dueto the presence of the streptavidin; in fact, the cylinders seemed tobecome more dispersed, which may have been due to the bound streptavidinacting like a steric stabilizer, keeping the cylinders isolated fromeach other. The solutions of sized vesicles also did not exhibit anyfeature resembling a vesosome. No LUVs were even present in thesesolutions.

EXAMPLE 2

[0087] A simple, one step process was developed to produce colloidalaggregates with a well defined size distribution by controlling theratio of reactive groups on the surface of the colloids (typicallyligands such as a biotin coupled to a phospholipid incorporated in avesicle membrane) to crosslinking agents (typically soluble biologicalreceptors such as avidin or streptavidin) in solution. Other chemicalligands associated with the colloidal particles, and covalentcrosslinking agents would also work as well. At a proscribed ratio ofligand to receptor, the receptor or crosslinker eventually saturates theligands at the colloid surface, thereby limiting the aggregationprocess. This limited aggregation process is initiated by simple mixingof the ligand-labeled colloidal particles with the crosslinking agent orreceptor. The crosslinking agent in solution competes for the limitednumber of surface ligands with ligands on other colloidal particles.

[0088] By having an excess of crosslinking agent, the ligands areeventually exhausted, and aggregation ceases when all of the ligands arecoupled to a crosslinker. The process requires no specific mechanical orphysical steps to initiate or limit the aggregation-aggregation proceedsby diffusion and reaction of the ligands and crosslinkers untilequilibrium (at least metastable equilibrium) is reached. A mathematicalmodel of the process was also developed that is consistent withexperiment and shows a well defined transition between completeflocculation and limited aggregation that depends primarily on the ratioof crosslinker to surface ligands. This process can be generalized toany system of colloidal particles with surface accessible, reactivegroups that could be coupled by a crosslinking agent.

[0089] The specific purpose of this embodiment is to have a one stepmethod of producing vesicle aggregates of a limited size or aggregationnumber for use in making the vesosome drug delivery system (S. A. Walkeret al., 1997). “Vesosomes” comprise a sized aggregate of unilamellarvesicles attached to each other via ligand-receptor interactions,encapsulated in a second bilayer, also attached via ligand-receptorinteractions (See FIG. 4). The interior vesicles can be of a single sizeand membrane or interior composition, or of varied sizes and/or membraneor interior composition. The exterior membrane may also be of differentcomposition, and may incorporate specific recognition or stericstabilization molecules on the surface.

[0090] For example, the total dimensions of a vesosome can be controlledfrom about 0.1 micron to >1 micron. The vesosome can incorporate avariety of water or lipid soluble drugs within the interior vesicles, orwithin the exterior capsule, or both. These drugs can then permeateslowly through the interior and exterior bilayers, providing acontrolled, slow release of drugs over time.

[0091] The one step process aggregation process replaces a more complex,multistep process that involve complete flocculation of the vesiclesfollowed by mechanical sizing via extrusion of the aggregated vesiclesthrough filters of defined size (S. A. Walker et al., 1997).

[0092] This process does not put any stress on the vesicles as theyaggregate nor does it require any additional filtering or purificationsteps as in the previous process. The filtering process also results indebris from destroyed vesicles and aggregates that needs to be removedprior to subsequent processing steps. Moreover, the surfaces of theselimited aggregates are saturated by the crosslinking agent, hence thesize distribution of the aggregates is stable for extended periods oftime. The entire process is completed in a few minutes and requires nosubsequent separations or purifications. The end result is a populationof well defined aggregates with surfaces saturated by streptavidin,avidin, or whatever crosslinking agent was used.

[0093] This process can be used much more generally to create a largercolloidal aggregate from small particles. The process is independent ofthe details of the colloidal particles, crosslinking agent, or surfaceassociated ligand. Prior to this work, non-specific colloidalaggregation induced by attractive interactions between the particlescould not typically be controlled, other than to completely inhibitaggregation by making the interaction between colloidal particlessufficiently repulsive. As discussed in detail in the later sections,once colloidal aggregation was initiated, the aggregates would growindefinitely and irreversibly. One of the only ways available to limitcoagulation of liquid colloidal droplets was to use a surface activecompound that could change the interaction between the colloidaldroplets as a function of surface coverage.

[0094] In what is generally referred to as limited coalescence, a fineemulsion of liquid droplets is generated whose surface area is muchlarger than can be completely covered by a surface stabilizing agent.These small droplets are unstable to coalescence and grow, with aconcomitant reduction in total interfacial area, until the stabilizingagent covers the interface at a sufficient level to halt furthercoalescence ( T. H. Whitesides and Ross (1995)).

[0095] The ideal construction process for a sub-micron bilayer baseddrug delivery system includes a series of equilibrium “self-assembly”steps that require only simple mixing and minimal equipment and minimalpurification. The main benefit of this new embodiment is to increase thespeed and efficiency of vesosome construction through (1) optimizing thevesicle aggregation process by creating a self-limiting, one-stepaggregation process by controlling the ratio of streptavidin to biotinand the total vesicle concentration described by theoretical models ofself-limiting vesicle aggregation. FIG. 4 shows an electron micrographof a vesosome constructed of 0.1 micron diameterdilaurylphosphatidylcholine interior vesicles aggregated via biotinatedlipids and streptavidin, encapsulated in a dioleoylphosphatidylserinebilayer, also coupled to the aggregate with the biotinatedlipid-streptavidin linkage. The overall dimensions of the vesosome isabout 0.5 microns. Increasing the efficiency of vesosome production is afirst step toward testing specific drug applications.

VESOSOME “CONSTRUCTION”

[0096] The vesosome can be designed to be sub-microscopic in size, withthe interior vesicles ranging from, for example, 20 -100 nm, and theentire aggregate from 0.1 to about 1 micron in diameter. FIG. 4 showsthat the vesosome contains aggregated, spherical, unilamellar vesiclessurrounded by an exterior membrane. The exterior membrane is continuousaround the aggregated vesicles and the size distribution is consistentwith that expected from the process described below.

[0097] The preparation of the vesosome is essentially a three-stepprocess. The first step is making the interior vesicles and loading thespecific drug to be delivered. These steps have been well worked out inthe literature (T. M. Allen et al., 1995; T. M. Allen, 1996; D. D.Lasic, 1993; D. D. Lasic et al., 1996). The second step is creating acontrolled-size vesicle aggregate, without disrupting the vesiclebilayer or contents. The third step is encapsulating the vesicleaggregate within an outer membrane.

[0098]FIG. 5 shows the process for creating a controlled size vesicleaggregate in Example 1 and an additional embodiment of “self-limiting”aggregation in this Example. In the process of Example 1, biotin-labeledvesicles were added to streptavidin solution, leading to completeflocculation of the vesicles via biotin-streptavidin-biotin crosslinks.These flocculated vesicles were then reduced in size mechanically byextrusion through filters of a given pore size. This step was followedby purification of the extrudate to remove debris and disruptedvesicles.

[0099] In the process of Example 2, a controlled ratio of streptavidinor avidin is added to the biotin labeled vesicles (or any crosslinkingagents), leading to aggregates of controlled size in a single mixingstep. No mechanical sizing is needed. The process of Example 2 providesfor the creation of aggregates that streamline vesosome production,eliminates time consuming mechanical filtration, separation, andextrusion steps, and helps to make the entire vesosome construction asimple series of controlled self-assemblies. These additionalself-assembly tools of self-limiting aggregation should also haveapplications well beyond vesosome production.

A New Self-Limiting Colloidal Aggregation Process

[0100] In the process of Example 1, as shown in FIG. 5, sufficientstreptavidin (Molecular Probes, Eugene, Oreg.) in buffer was added tovesicles containing a small fraction of biotin-lipid (Biotin-X-DPPE,Molecular Probes) to produce an overall streptavidin to biotin-lipidratio of 1:15; however, the ratio of streptavidin to biotin-lipid on theoutside of the vesicle available for binding was approximately 1:8. Theremainder of the vesicle bilayer composition could be varied betweenpure dioleyolyphosphatidylcholine to mixtures ofdistearoylphosphatidylcholine and cholesterol and did not affect theresults of the aggregation process. As streptavidin has four distinctbinding sites for biotin, the ratio of streptavidin binding sites toexposed biotin was 1:2, meaning there are always unreactedbiotin-lipids. Within an hour after adding the streptavidin to thevesicle solution, the suspension changed from clear and bluish to opaqueand cloudy-white, indicating that vesicle aggregates were forming.Aggregation continued indefinitely, producing multi-micron sizedaggregates that eventually flocculated (S. A. Walker et al., 1997; T. H.Whitesides et al., 1995; T. M. Allen et al., 1995; T. M. Allen, 1996; D.D. Lasic, 1993; D. D. Lasic et al., 1996; S. Chiruvolu et al., 1994).However, aggregates for intravenous use must be of order 0.2-0.5 micronsto facilitate long circulation times (with steric stabilization byPEG-lipid (T. M. Allen, 1996; D. D. Lasic, 1993; D. D. Lasic et al.,1996)). In the process of Example 1, the large vesicle aggregates wereextruded through two stacked Nuclepore filters of pore size 1 μm. Thisproduced a dispersion of vesicle aggregates with sizes ranging from0.3-1.0 μm. The result that was that there was a large fraction ofisolated vesicles and much smaller aggregates that would have to beremoved at this step.

[0101] A simple, one-step, self-limiting aggregation process couldsignificantly increase both the efficiency and speed of vesosomeconstruction. However, colloidal aggregation is typically an all ornothing process when the interactions leading to the aggregation areattractive, but non-specific. However, we have found that if we increasethe ratio of streptavidin to biotin so that there is roughly two biotinlipid sites available on the vesicle surface per streptavidin added(1:2) (experimentally, this corresponds to an initial mole ratio ofroughly 4 biotin lipids per streptavidin, as half of the biotins pointtoward the interior of the vesicles, where they are not available forcross-linking), the aggregation process appears to be self-limiting.That is, the aggregation process stops with finite sized aggregates thatare stable (See FIG. 6).

Modified Smolukowski Equation for Aggregation

[0102] In the original process described in Example 1 (S. A. Walker etal., 1997; T. H. Whitesides et al., 1995; T. M. Allen et al., 1995; T.M. Allen, 1996; D. D. Lasic, 1993; D. D. Lasic et al., 1996; S.Chiruvolu et al., 1994), vesicles (0.1 micron diameter) incorporating asmall fraction of biotin-lipid could be completely aggregated whensufficient streptavidin or avidin (Molecular Probes) was added toproduce a streptavidin to exposed biotin-lipid mole ratio, R, ofapproximately 1:8.

[0103] Titration of vesicles incorporating 0.16 mol % of biotin-X DHPEwith fluorescent BODIPY-labeled avidin or streptavidin (MolecularProbes, Eugene, Oreg.) showed that the fluorescence intensity increasedlinearly up to a streptavidin to total biotin-lipid mole ratio between1:8 and 1:9 at which the fluorescence saturated. As streptavidin has 4binding sites per molecule, this shows that roughly one half of thetotal biotin-lipids were exposed on the outside of the vesicle. This isconsistent with the expected complete miscibility of the biotin-X DHPEwith the vesicle phospholipids.

[0104] As streptavidin (or avidin) has four distinct binding sites forbiotin, there were always unreacted biotin-lipids exposed on the vesiclesurface. Within a few minutes after adding the streptavidin to thevesicle solution, the suspension changed from clear and bluish to opaqueand cloudy-white, indicating that vesicle aggregates were forming.Aggregation continued indefinitely, producing multi-micron sizedaggregates that flocculated (S. A. Walker et al., 1997; T. H. Whitesideset al., 1995; T. M. Allen et al., 1995; T. M. Allen, 1996; D. D. Lasic,1993; D. D. Lasic et al., 1996; S. Chiruvolu et al., 1994) (FIG. 7).

[0105] However, as the ratio, R, of streptavidin to exposed biotin-lipidwas increased to one streptavidin to less than four biotin-lipidsavailable on the vesicle surface (R≦1:4), aggregation began to diminishas shown by dynamic light (DLS) scattering (FIG. 7). As the streptavidinto exposed biotin-lipid ratio was further decreased, (R≧1:2)flocculation ceased and DLS showed a dramatic decrease in the averageaggregate size. This was confirmed by freeze-fracture electronmicroscopy (J. A. Zasadzinski et al., 1989) that showed a stabledistribution of aggregates about 0.5 microns in diameter formed from the0.1 micron diameter vesicles (FIG. 6). For larger values of R, theextent of aggregation did not change appreciably with R. No significantdeformation of the vesicles occurred during any of the aggregationprocesses as shown by similar releases of entrapped carboxyfluoresceindye from aggregated and unaggregated vesicles.

[0106] While previous experiments showed that excess biotin-lipid led tocomplete aggregation (S. A. Walker et al., 1997; T. H. Whitesides etal., 1995; T. M. Allen et al., 1995; T. M. Allen, 1996; D. D. Lasic,1993; D. D. Lasic et al., 1996; S. Chiruvolu et al., 1994), and a largeexcess of streptavidin led to very limited aggregation (H. C. Loughreyet al., 1990), the dramatic transition with receptor-ligand ratio wassurprising. Vesicles aggregate by coupling a biotin-lipid on one vesicleto a streptavidin bound to a biotin-lipid on a second vesicle. Theinitial step in this process in the binding of a streptavidin insolution to the biotin lipid on a given vesicle. A competition foravailable biotin sites is set up between free streptavidin in solutionand streptavidin already bound to another vesicle. Hence, theaggregation process is both initiated and inhibited by free receptor insolution. Sufficient streptavidin in solution eventually leads to thesaturation of the ligands on the surface of the growing aggregate. Onceall of the biotin-lipid sites on the growing vesicle aggregates aresaturated with streptavidin, aggregation ends, leaving finite sizedaggregates.

[0107] The classical description of rapid aggregation of colloidalparticles is given by the Smolukowski equation, which has been shown togive reasonable agreement with experiment for non-specificdiffusion-controlled colloidal aggregation (D. F. Evans et al., 1994).The Smolukowski equation gives the diffusion controlled rate ofproduction of aggregates of size j and concentration [P_(j)] fromsmaller aggregates (i<j), less the consumption of aggregates of size jby further aggregation with any other aggregate. The rate constant, k isgiven by the mutual diffusion of the particles toward each other and isassumed to be constant, independent of the size of the particles or theaggregates: $\begin{matrix}{{{d\quad\left\lbrack P_{j} \right\rbrack}/{dt}} = {k\quad\left\lbrack {{{1/2}{\sum\limits_{i > j}{\left\lbrack P_{i} \right\rbrack \quad\left\lbrack P_{j - i} \right\rbrack}}},{{- \left\lbrack P_{j} \right\rbrack}{\sum\limits_{i}\left\lbrack P_{i} \right\rbrack}}} \right\rbrack}} & (1)\end{matrix}$

[0108] and the change in the total particle concentration,$\sum\limits_{i}\left\lbrack P_{j} \right\rbrack$

[0109] is: $\begin{matrix}{{{d/{dt}}{\sum\limits_{j}\quad \left\lbrack P_{j} \right\rbrack}} = {{{- k}/2}\left( {\sum\limits_{j}\left\lbrack P_{j} \right\rbrack} \right)^{2}}} & (2)\end{matrix}$

[0110] For an initial monomer (vesicle) concentration, [P₀], at t=0,Eqn. 3 has the solution: $\begin{matrix}{{\sum\limits_{j}\left\lbrack P_{j} \right\rbrack} = {{\left\lbrack P_{o} \right\rbrack/1} + {t/\tau}}} & (3)\end{matrix}$

[0111] in which τ=2/k[P₀]. The mean aggregation number, M, diverges forlong times, resulting in flocculation of the colloidal particles:$\begin{matrix}{M = {{\left\lbrack P_{o} \right\rbrack/{\sum\limits_{j}\left\lbrack P_{j} \right\rbrack}} = {1 + {t/\tau}}}} & (4)\end{matrix}$

[0112] The diffusion limited rate constant, k_(ij), is given by themutual diffusion of the particles toward each other:k_(ij)=2k_(B)T/3_(η()1/R_(i)+1/R_(j))(R_(i)+R_(j)). For the limitingcase of R_(i)=R_(j), k_(ij)=k=8k_(B)T/3η=8×10⁹ liter/mol-sec, in whichk_(B) is Boltzman's constant, T is absolute temperature, and η is thesolvent viscosity. For ligand-receptor induced aggregation, a much lowerrate constant than diffusion limited is expected due to the stericrequirements of the ligand-receptor bond.

Self-Limiting Aggregation

[0113] However, these expressions do not describe aggregation caused bycross-linking a limited number of reactive sites on the colloidsurfaces. Biotin-lipids on different vesicles must be crosslinked bystreptavidin to induce aggregation. If θ is the average fraction ofbiotin-lipids bound to streptavidin, a vesicle with θ>0 must contact avesicle with free biotin-lipid, (1−θ0)>0, in order for the vesicles tobind. The new expression for the change of total particle concentrationis (See Eqns. 3, 4): $\begin{matrix}{{{d/{dt}}{\sum\limits_{j}\left\lbrack P_{j} \right\rbrack}} = {{{- k}/2}\left( {\theta \left( {1 - \theta} \right)} \right)\left( {\sum\limits_{j}\left\lbrack P_{j} \right\rbrack} \right)^{2}}} & (5)\end{matrix}$

[0114] As θ goes from zero to one, the rate of aggregation goes througha maximum, then decreases and eventually stops, giving a finite numberof aggregates: $\begin{matrix}{{\sum\limits_{j}\left\lbrack P_{j} \right\rbrack} = {{\left\lbrack P_{o} \right\rbrack/1} + \left\lbrack {\left( {\int_{0}^{t}{{\theta \left( {1 - \theta} \right)}{t}}} \right)/\tau} \right\rbrack}} & (6)\end{matrix}$

[0115] with a finite aggregate size, M: $\begin{matrix}{M = {1 + \left\lbrack {\left( {\int_{0}^{\infty}{{\theta \left( {1 - \theta} \right)}{t}}} \right)/\tau} \right\rbrack}} & (7)\end{matrix}$

[0116] again, τ=2/k[P₀]. The average particle size depends on the timeevolution of the bound biotin fraction, θ, which in turn is coupled tothe size distribution, [P_(j)].

[0117] Each different size aggregate will likely have a differentfraction of biotin lipid coupled to streptavidin, θ_(j). If all theθ_(j) are set equal (in the same level of approximation as the originalSmolukowski equation) the equations are greatly simplified and ananalytical solution is possible.

[0118] However, it is possible to write a simplified equation for θ thatreflects the initial competition for biotin sites on the unaggregatedvesicles, and thereby decouple the expressions for [P_(j)] and θ. Thefirst term in Eqn. 8 is a simple binary expression for reaction of thebiotin sites with streptavidin in solution.

[0119] Diffusion and reaction of biotin-lipid with a biotin-lipidattached to streptavidin on a given vesicle also leads to an increase inθ. Biotin-lipid and/or biotin-lipid attached to a streptavidin will alsodiffuse towards existing contact sites between vesicles. At thesecontact sites, multiple bonds between a vesicle pair can form, leadingto a depletion of free biotin (D. Leckband et al., 1995). In Eqn. 8,these effects have the same form as the second term of Eqn. 8, with k₂being replaced by an effective rate constant that reflects all threepossible effects. As k₂ increases relative to k₁, δ decreases relativeto R (Eqn. 10), and θ−>1 faster (Eqn. 11). If the vesicle suspension issufficiently dilute, complete aggregation does not occur for any valueof R, and there is no threshold. For our experiments, this occurred forvesicle concentrations ≦1 mg/ml.

[0120] The second term is the crosslinking of a streptavidin occupiedsite on one vesicle with a free biotin site on a second vesicle:$\begin{matrix}{{{n\quad\left\lbrack P_{o} \right\rbrack}\frac{\theta}{t}} = {{k_{1}{n\quad\left\lbrack P_{o} \right\rbrack}\quad \left( {1 - \theta} \right)N_{s}} + {k_{2}\quad \left( {n\quad\left\lbrack P_{o} \right\rbrack} \right)^{2}{\theta \left( {1 - \theta} \right)}}}} & (8)\end{matrix}$

[0121] n is the number of exposed biotin sites per vesicle; the vesiclesare at an initial concentration of [P₀] Hence, n[P₀] is the totalbiotin-lipid concentration exposed on the surface of the vesicles.

[0122] Titration of vesicles incorporating 0.16 mol % of biotin-X DHPEwith fluorescent BODIPY-labeled avidin or streptavidin (MolecularProbes, Eugene, Oreg.) showed that the fluorescence intensity increasedlinearly up to a streptavidin to total biotin-lipid mole ratio between1:8 and 1:9 at which the fluorescence saturated. As streptavidin has 4binding sites per molecule, this shows that roughly one half of thetotal biotin-lipids were exposed on the outside of the vesicle. This isconsistent with the expected complete miscibility of the biotin-X DHPEwith the vesicle phospholipids.

[0123] N_(S) is the concentration of streptavidin in solution:

N _(S) =N _(SO) −βn[P _(O)]θ  (9)

[0124] N_(S,O) is the initial streptavidin concentration and β is ratioof streptavidin to bound biotin. β varies from 1, which corresponds toonly one of the binding sites of streptavidin being full, to ¼, whichcorresponds to all four streptavidin sites being bound to biotin: ¼≦β≦1.To decouple the equations, it is necessary to make β constant.

[0125] β must start our equal to 1, then decrease to a lower value thatlikely depends on the streptavidin to biotin ratio. However, goodagreement with the fluorescence data. (FIG. 6) is obtained with δtreated as a fitting parameter, suggesting that β approaches a steadystate value.

[0126] Inserting Eqn. 9 into Eqn. 8, we have, with R=N_(S,O)/n[P_(O)] asthe initial ratio of streptavidin to exposed biotin-lipids:$\begin{matrix}{{\frac{\theta}{t} = {{n\quad\left\lbrack P_{o} \right\rbrack}{k_{1}\left( {R - {\delta\theta}} \right)}\quad \left( {1 - \theta} \right)}}{\delta = {\beta - \frac{k_{2}}{k_{1}}}}} & (10)\end{matrix}$

[0127] The solution for θ has the following form: $\begin{matrix}{\theta = \frac{{\exp \quad\left\lbrack {\left( {1 - \frac{\delta}{R}} \right)\quad \frac{t}{\tau_{1}}} \right\rbrack} - 1}{{\exp \quad\left\lbrack {\left( {1 - \frac{\delta}{R}} \right)\quad \frac{t}{\tau_{1}}} \right\rbrack} - \frac{\delta}{R}}} & (11)\end{matrix}$

[0128] τ₁=1/k₁N_(S,O), the time constant for streptavidin addition tobiotin-lipids. For δ/R<1, for long times (t−>∞), θ−>1 and the outervesicle surface is saturated by streptavidin. For δ/R>1,θ−>R/δ, andthere are always unreacted biotin-lipids on the vesicle surface.Inserting Eqn. 11 into Eqn. 7, for δ/R<1, gives the mean aggregate sizeat equilibrium: $\begin{matrix}{M = {1 + {\frac{\tau_{1}}{\tau}{\left( \frac{R}{\delta} \right)^{2}\left\lbrack {{- \left( \frac{\delta}{R} \right)} - {\ln \quad \left( {1 - \frac{\delta}{R}} \right)}} \right\rbrack}}}} & (12)\end{matrix}$

[0129] M diverges for δR≧1. Eqn 12 gives a very good representation ofthe DLS data in FIG. 7. From FIG. 7, the extent of aggregation isindependent of vesicle and streptavidin concentration, and the criticalvalue of R when the aggregate size diverges (corresponding to δ/R=1 inEqn. 12), is R_(crit)≈0.3=δ_(crit).

[0130] Diffusion and reaction of biotin-lipid with a biotin-lipidattached to streptavidin on a given vesicle also leads to an increase inθ. Biotin-lipid and/or biotin-lipid attached to a streptavidin will alsodiffuse towards existing contact sites between vesicles. At thesecontact sites, multiple bonds between a vesicle pair can form, leadingto a depletion of free biotin (D. Leckband et al., 1995). In Eqn. 8,these effects have the same form as the second term of Eqn. 8, with k₂being replaced by an effective rate constant that reflects all threepossible effects. As k₂ increases relative to k₁, δ decreases relativeto R (Eqn. 10), and θ−>1 faster (Eqn. 11). If the vesicle suspension issufficiently dilute, complete aggregation does not occur for any valueof R, and there is no threshold. For our experiments, this occurred forvesicle concentrations ≦1 mg/ml (D. A. Noppl-Simson et al., 1996).

[0131] The model can be further evaluated by monitoring the timedependence of the fluorescence of BODIPY-labeled streptavidin as itbinds to the biotin-lipids. The fluorescence of the labeled streptavidinis linearly proportional to the number of biotins bound to thestreptavidin; hence, this is a direct measure of θ, the average fractionof bound biotin-lipids (N. Emans et al., 1995). The fluorescenceintensity as a function of time was measured for a fixed BODIPY-labeledstreptavidin concentration (N_(S,O) constant in Eqns. 10-12) whendifferent concentrations of 0.1 micron vesicles of DLPC vesiclesincorporating 0.16 mole % of biotin-X DHPC were added and allowed toaggregate. The fit of this data to Eqn. 12 for all of the ratios wassurprisingly good considering the limitation of the model. Averagingfrom the fits, τ₁=1/k₁N_(S,O), which should be constant between theexperiments, is ≈700±100 sec; hence k₁≈4×10⁴ liter/mol-sec. Thediffusion limited rate constant, k_(ij), is given by the mutualdiffusion of the particles toward each other:k_(ij)=2k_(B)T/3η(1/R₁+1/R_(j))(R_(i)+R_(j)). For the limiting case ofR_(i)=R_(j), k_(ij)=k=8k_(B)T/3η=8×10⁹ liter/mol-sec, in which kB isBoltzman's constant, T is absolute temperature, and η is the solventviscosity. For ligand-receptor induced aggregation, a much lower rateconstant than diffusion limited is expected due to the stericrequirements of the ligand-receptor bond.

[0132] The second parameter, δ, increases as R increases, from about 0.2at R=0.125 to about 0.3 for R=0.5 to nearly 1 when R=4, but more slowlythan R, leading to the crossover between complete flocculation (δ/R>1)to self-limited aggregation (δ/R<1). The increasing value of δ suggeststhat that the average number of streptavidins per bound biotin-lipids,β, in Eqn. 11, increases as R increases, which is consistent withsaturation of the vesicle surfaces with streptavidin. The competitionfor the biotin-lipids at the vesicle surface appears to be the cause ofthe percolation-like behavior.

[0133] To summarize, the extent of ligand-receptor induced vesicleaggregation can be controlled by varying the ratio of soluble receptorto surface-bound ligands. Aggregation exhibits a dramatic change withthis ratio—below a critical value, aggregation is self-limiting, theaggregation numbers are finite, and the aggregates remain suspended insolution. Above this critical value, aggregation is complete and theaggregates grow indefinitely and flocculate. A biological system couldbe controlled to exist near this percolation threshold so that onlysmall perturbations would cause the system to cross-over. The thresholdcould also be crossed by altering the number of binding sites on thereceptor, or by altering the long-range forces between the ligands andreceptors (D. Leckband, 1995; D. E. Leckband et al., 1994; D. Leckbandet al., 1995), between the receptors and vesicles, or between thevesicles themselves (S. A. Walker et al., 1997). This type of reactioninduced aggregation could also be generalized to other colloidal systemsby incorporating a competitive cross-linking reaction at the colloidsurface and would be a useful new way to controllably alter the sizedistribution of a colloidal dispersion.

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What is claimed is:
 1. A composition comprising a bilayer structure andmultiple containment units, the bilayer structure encapsulating themultiple containment units.
 2. The composition of claim 1 , wherein thecontainment units are aggregated within the bilayer structure.
 3. Thecomposition of claim 1 , wherein the multiple containment units includea therapeutic agent
 4. The composition of claim 1 , wherein the multiplecontainment units include a diagnostic agent.
 5. A vesosome having abilayer structure and multiple vesicles, the bilayer structureencapsulating the multiple vesicles.